miRCURY LNA™ microRNA Array Kit - Exiqon · Hybridization and washing using an Agilent hybridization SureHyb ... miRCURY LNA™ microRNA Power Labeling Kit or miRCURY LNA™ microRNA

Instruction manual v2.1for product # 208400, 208401, 208402, 208420, 208421, 208422March 2011

miRCURY LNA™ microRNA Array Kit6th generation - human, mouse & rat

The 6th GenArray layout

has newdimensions

2

mIC

Ro

RN

A AR

RAY K

IT | Instruction Manual

Table of Contents

Product Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4miRCURY LNA™ microRNA Array Kit content . . . . . . . . . . . . . . . . . . . . . . . 4Additional required material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4miRCURY LNA™ microRNA Array layout . . . . . . . . . . . . . . . . . . . . . . . . . . . 5Array probes specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6How to use the Spike-in microRNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8Scanner settings and array orientation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Protocol overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12Experimental workflow overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12Single or dual color . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Labeling Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15Protocol & Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Hybridization Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19Hybridization and washing using Tecan HS Pro™ hybridization stations 19Hybridization and washing using an Agilent hybridization SureHyb chamber kit and gasket slide kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22Hybridization and washing using NimbleGen/ MAUI® 4-Bay or 12-Bay hybridization stations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Recommendations for Experimental Set-up and Data Handling . . . . . . . . . 33Software and Databases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

ImaGene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37Nexus Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37Links to other array software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

GenePixw Array List (GAL) files can be found at www .exiqon .com/Gal-downloads

3

mIC

Ro

RN

A AR

RAY K


GenePix® Array List (GAL) files can be found at www .exiqon .com/Gal-downloads

Tips and Trouble Shooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39Experimental procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Preparation of RNA sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Black spots - purification of the labeling reaction I . . . . . . . . . . . . . . . 40 Concentration and purification of the labeling reaction II . . . . . . . . . . 40 Solid particles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 Flushing the hybridization chamber . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 Dry slides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 No signals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 High signals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 High background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42Spike-in miRNA kit signal distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 Sample signal distribution for Spike-in miRNA Kit v2 (product # 208041) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 Sample signal distribution for Spike-in miRNA Kit (product # 208040) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45Related Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

4

mIC

Ro

RN

A AR

RAY K


Product Summary

miRCURY LNA™ microRNA Array Kit content

microarray slidesmiRCURY LNA™ microRNA Arrays consist of control probes, and approximately 2383 capture probes, complementary to human, mouse, rat, and their related viral sequences from the v .16 .0 release of miRBase . Arrays also contain a number of proprietary human miRPlus™ sequences not yet in miRBase .

Hybridization buffer (product # 208022)Products with 3, 6 and 24 slides: 1 bottle x 5 mL

20x Salt buffer (product # 208023)Products with 3 and 6 slides: 1 bottle x 125 mLProducts with 24 slides: 2 bottles x 125 mL

10% Detergent solution (product # 208024)Products with 3 and 6 slides: 1 bottle x 15 mLProducts with 24 slides: 2 bottles x 15 mL

Spike-in microRNA kit v2 (product# 208041)52 synthetic 5’-phosphorylated microRNAs, dried-down, 2x 24 reactions

Additional required material

miRCURY LNA™ microRNA Power Labeling Kit or miRCURY LNA™ microRNA Hi-Power Labeling KitFluorescent labeling of microRNAs from total RNA samples ready for hybridization on arrays (product # 208030-A, 208031-A, 208032-A, 208033,208034, 208035) .

For manual hybridizationMicroarray Hybridization Chamber - SureHyb (Agilent product# G2534A)Hybridization Gasket Slide Kit (Agilent product# G2534-60003)Hybridization oven with rotation . Glass staining jar/dish or equivalent .

5

mIC

Ro

RN

A AR

RAY K


miRCURY LNA™ microRNA Array layout

The array is located on a standard-sized slide as illustrated in the drawing below (25 .4 mm by 76 .2 mm or 1 in by 3 in) . The array carries the following specifications:• Outer dimensions: 13 mm wide by 54 mm long• 36 sub-arrays• Spot size: 100 µm• Distance between spots: 215 µm

Figure 1

To assist in orientation of the array and positioning of the image analysis grid, Hy3™ fluorescent labeled “landing lights” are present in all 4 corners plus one extra in lower right corner of the 36 sub-arrays, 180 total . The slides are com -patible with all major brands of microarray scanners, that take glass slides of the above described specifications (1 x 3 inches, not Affymetrix and Illumina scanners) .

The miRCURY LNA™ microRNA Array, REV (Figure 1, bottom image) which is accommodating MAUI®/NimbleGen™ mixers and hybridization stations is printed on the opposite side of the barcode .

8 mm

13 mm

54 mm

6,1 mm

14 mm Product # 208400208401and208402

8 mm

Product # 208420208421and208422

13 mm

54 mm

6,1 mm

14 mm

6

mIC

Ro

RN

A AR

RAY K


Array probes specifications

Tm-normalized capture probesThe miRCURY LNA™ microRNA Array slides contain capture probes complementary to mature microRNAs registered in miRBase . The capture probes are Locked Nucleic Acid (LNA™) enhanced oligonucleotides . By varying the LNA™ content and length, the capture probes are Tm-normalized to hybridise optimally under the conditions described in this protocol .

Coverage of probe set The slides contains capture probes for all microRNAs in human, mouse, rat and their related viruses as annotated in miRBase Release 16 .0 . Please go to www .exiqon .com/array to see the coverage in respect to latest version of miRBase . In addition, a number of capture probes are available for detection of microRNAs not included in miRBase . These miRPlus™ probes give researchers access to information unavailable elsewhere .Please go to our online microRNA resource at www .exiqon .com/gal-downloads to:• Download species-specific GenePix® Array Lists (GAL) files, consistent with

the latest updates to miRBase . Please note the product number and lot# on the array slide box and on the slide pouch . This number is needed to identify the GAL file .

Control capture probesA number of control capture probes are included in the probe set . • Spike-in control probes to ensure optimal labeling and hybridization . • Negative control capture probes .• Capture probes complementary to small nuclear RNAs .Please see table on the next page for details .

7

mIC

Ro

RN

A AR

RAY K


List of control probes

Probe ID Positive controls Aliases Validated in these organisms

46202 5S_rRNA - hsa145659 RNU1 U1; HSD1; RNU1; U1A1; HU1-1;

RNU1A; RNU1A3; RNU1G4; Rnu1a1hsa, mmu

145657 RNU5 U5a; Rnu5a hsa, mmu11278 RNU6-1 U6; RNU6; RNU6A hsa, mmu, rno11279 RNU6-1/RNU6-2 U6; RNU6; RNU6A / U6; RNU6B hsa, mmu, rno19011 SNORD10 mgU6-77 hsa145661 SNORD110 HBII-55 mmu19005 SNORD118 U8 hsa19606 SNORD12 HBII-99 hsa19603 SNORD13 U13 hsa19013 SNORD14B U14; U14B; RNU14B hsa19607 SNORD15A U15A; RNU15A hsa19008 SNORD2 R39B; SNR39B hsa19007 SNORD3@ U3; U3A; U3B hsa46204 SNORD38B U38B; RNU38B hsa46206 SNORD44 U44; RNU44 hsa46205 SNORD48 U48; RNU48 hsa46203 SNORD49A U49; U49A; RNU49 hsa19604 SNORD4A Z17A; RNU101A; mgh18S-121 hsa19605 SNORD6 mgh28S-2412 hsa

145663 SNORD65 HBII-135 mmu46197 SNORA66 HBII-142 hsa

145666 SNORD68 HBII-202 mmu

Probe ID Negative controls Validated negative control in these organisms

14258 hsa_negative_control-1 hsa, mmu, rno

14259 hsa_negative_control-2 hsa






List of control probes: The different control capture probes were compared against the genomic sequence of hsa, mmu and rno, with the BLAST tools at www .ensembl .org . The right column show species for which the positive control probes have a perfect sequence match . Negative probes are listed with species for which they are designed as mismatch sequences .

8

mIC

Ro

RN

A AR

RAY K


Note

In the GAL-file, only capture probes relevant to the species in question are annotated with a name . Probes that do not have a name could be designed for another species, internal controls or obsolete probes no longer in use . Some of these may show signal although they are not annotated, but they should be ignored in the analysis .

Some capture probes have been optimized from previous versions of the miRCURY LNA™ Array . These will appear with a new probe ID on this array compared to earlier versions . For more details about comparisons to older versions of the arrays, please contact www .exiqon .com/contact .

How to use the Spike-in microRNAs

The miRCURY LNA™ microRNA Array Spike-in kit v2 contains 52 different synthetic unlabeled microRNAs in different concentrations . The set can be spiked into an RNA sample prior to labeling and the synthetic Spike-in kit v2 will hybridize to corresponding capture probes on the miRCURY LNA™ microRNA Array . Spike-in microRNAs added in equal amounts to the different labeling reactions before hybridization can be used to improve the data quality as control for the labeling reaction or in calibrating the scanner settings as well as for normalization and assessing the technical variability between different part of the array experiment: • If the variation between replicates of each of the four spike-in controls exceed 20-25%, it could be an indication of insufficient agitation of the sample .• Inter- as well as intra correlations between all 52 spike-in miRNAs are normally within 0 .950 and 0 .999 (R2) .

The Spike-in miRNA kit v2 has been designed and tested not to cross-react with endogenous microRNAs from human, mouse or rat, and is provided at concentrations compatible with endogenous microRNA expression levels . The Spike-in kit v2 is supplied with different concentrations of synthetic spike-in microRNAs aimed at spanning the whole intensity range of microRNAs in most tissue samples .

9

mIC

Ro

RN

A AR

RAY K


Each spike-in miRNA has 4 replicates of capture probes on the array . Four of the capture probes (ID # 27968, 28568, 28684 and 29001) are printed in two diagonals in each of the 36 subgrids (see figure 4) . A smaller spike-in miRNA kit (product # 208040) is also available for the potential use of hybridization control . See signal distribution details for both kits in the tips section .

List of Spike-in miRNA kit v2 capture probes and their probe ID’s:* Indicates that these probes are printed in two diagonals in all subgrids . 72 + 4 replicates in total (see tip 12 for layout overview) . The different control and spike-in miRNA capture probes were compared against the genomic sequence of hsa, mmu and rno, with the BLAST tools at www .ensembl .org .

See tip 10-11

Probe ID Name1100 spike_control_v2_113186 spike_control_v2_213367 spike_control_v2_313371 spike_control_v2_413388 spike_control_v2_513389 spike_control_v2_613393 spike_control_v2_713417 spike_control_v2_813421 spike_control_v2_913430 spike_control_v2_1024127 spike_control_v2_1124136 spike_control_v2_1224163 spike_control_v2_1324199 spike_control_v2_1424217 spike_control_v2_1524226 spike_control_v2_1625557 spike_control_v2_1725593 spike_control_v2_1825611 spike_control_v2_1925728 spike_control_v2_2026160 spike_control_v2_2127291 spike_control_v2_2227318 spike_control_v2_2327350 spike_control_v2_2427676 spike_control_v2_2527821 spike_control_v2_26

Probe ID Name27833 spike_control_v2_2727953 spike_control_v2_2827968 spike_control_v2_29*28038 spike_control_v2_3028098 spike_control_v2_3128393 spike_control_v2_3228444 spike_control_v2_3328488 spike_control_v2_3428568 spike_control_v2_35*28581 spike_control_v2_3628684 spike_control_v2_37*28876 spike_control_v2_3828929 spike_control_v2_3929001 spike_control_v2_40*29056 spike_control_v2_4129138 spike_control_v2_4229146 spike_control_v2_4329544 spike_control_v2_4429564 spike_control_v2_4529837 spike_control_v2_4630147 spike_control_v2_4730207 spike_control_v2_4830293 spike_control_v2_4930747 spike_control_v2_5030756 spike_control_v2_5132812 spike_control_v2_52

List of capture probes for Spike-in miRNA Kit v2

10

mIC

Ro

RN

A AR

RAY K


Scanner settings and array orientation

4 of the spike-in control probes (marked with * on page 9) are located in two diagonals, each in a total of 72 replicas . When scanning the images, these spike-in capture probes can be used to determine appropriate scanner settings . If spike-in mix is added in equal amounts to both RNA samples, the signal from the spike-in capture probes should be similar in both channels after scanning for dual color experiments . First find a laser power setting that gives the expected signal range and then adjust PMT settings so that both channels give similar signal in the spike-in capture probes . The landing lights (annotated as Hy3™ in the gal-file, probe ID 13138) should not be used for finding the proper scanner settings as these spots contain dye spotted directly on the arrays . The intensity of these spots may vary from batch to batch of slides . These spots are only included for gal-file orientation, and their corresponding data points should be removed prior to normalization of the dataset .

Figure 2. The location of spike-in capture probes in each subgrid on miRCURY LNA™ Arrays . The green spots are the Hy3 landing lights . The red spots in the diagonal are probe # 29001, 28684, 28568 and 27968 counting from upper left, targeting complementary RNA’s in the Spike-in miRNA v2 kit .

Std probes (4 replicates)Landing lightControl probes (replicatesin all 72 subgrids)

Figure 2

11

mIC

Ro

RN

A AR

RAY K


Storage

miRCURY LNA™ microRNA Arrays should be stored desiccated at room temperature and protected from light . When properly stored, arrays will remain hybridization competent for at least 12 months from production date . The arrays are shipped in desiccated, re-sealable storage pouches that are ideal for this purpose . Both the storage pouches and slide storage boxes are manufactured from materials that minimize outgassing and effectively block exposure to foreign contaminants that can elevate background levels . When accessing arrays, remove only the arrays you plan to immediately use and return the remaining arrays to the pouch quickly, leaving the desiccant pack in place .

Dissolve the miRCURY LNA™ Array Spike-in miRNA Kit v2s in 30 µL/vial (see important note page 16) of RNase free water (supplied) upon receipt . Vortex to thoroughly dissolve the lyophilized RNA, pulse briefly in a microfuge, and leave the suspension on ice for 30 min . to dissolve . Vortex and then spin to collect tube contents . Store the dissolved Spike-in microRNA at –20° C until use and avoid repeated cycles of freeze/thawing . You may wish to aliquot the dissolved spike-in microRNAs to avoid repeated freeze/thawing . For long-term storage, keep the vial at –80° C .If stored properly, shelf life for the miRCURY LNA™ Array Spike-in miRNA kit v2s is 12 months . In solution, the shelf life for the spike-in microRNAs is 3 months .

12

mIC

Ro

RN

A AR

RAY K


CIP treatment Mix: RNA sample, CIP buffer, Spike-In microRNA and CIP enzyme

Labeling reaction Mix: CIP treated RNA sample Labeling buffer Hy3™ or Hy5™ DMSO Enzyme

miRCURY LNA™ microRNA Hi-Power Labeling Kit

Mix samples Dual color mix: Hy3™ labeled sample Hy5™ labeled sample Hybridization bufferDenature sample

Single color mix: Hy3™ labeled sample Hybridization bufferDenature sample

miRCURY LNA™ microRNA Array Kit

Hybridize Hybridize at 56°C for 16 hours

Stringency wash Wash 2 min . in buffer A at 56°CWash 2 min . in buffer B at 23°CWash 2 min . in buffer C at 23°CDry slides

Image acquisition Scan slides (recommended scan at 10µm)Download relevant GAL files from www .exiqon .com/gal-downloads

Protocol overview

Workflow for the complete microRNA array experiment combining the sample labeling and array hybridization protocol

13

mIC

Ro

RN

A AR

RAY K

IT | Instruction ManualAdvantages:

• Lowess normalization reduces differences caused by experimental variation

• Lowess normalization reduces day to day variation

• Ratio data are typically more robust than absolute signals

Disadvantages:• With common reference:

Can only be done on a limited project with known number of samples and requires double the amount of RNA .

• With universal reference: miRs expressed in samples but not in reference are measured inaccurately .

• Hy5™ is sensitive to ozone and might pose problems especially in urban areas if counter measures have not been taken in the lab to ensure low ozone levels

• Requires double the amount of RNA sample than single color

Data analysis:• Local background

subtraction (or norm exp)

• Lowess normalization

Arrays can either be run as dual color (Hy3™ vs Hy5™) or single color (Hy3™ only) . A schematic overview of advantages and disadvantages as well as recommended data analysis method can be seen in this table:

Tip: For a visual presentation of the labeling procedure and a manual hybridization, please view the instructional movie at www .exiqon .com/e-talk

Single or dual color

Dual color

14

mIC

Ro

RN

A AR

RAY K


*Single color is only supported if the RNA is labeled using the miRCURY LNA™ microRNA Array Hi-Power labeling kit or miRCURY LNA™ microRNA ArrayPower labeling kit, and Exiqons buffers etc .

Experimental designIf running single color, each sample has to be labeled with Hy3™, and hybridized to individual arrays . If running dual color, it is recommended that each sample is labeled with Hy3™ and that the reference is labeled with Hy5™ . As reference we recommend to use either a common reference or a universal reference .

Advantages:• Enables comparison

across experiments• Ability to add more

samples to an experiment later

Disadvantages:• Requires extremely high

lab standards and very reproducible handling of samples

• Experiments performed over a large time span are sensitive to minor lot to lot variations

Data analysis:• Local background

subtraction (or norm exp)

• Quantile normalization

Single color*

15

mIC

Ro

RN

A AR

RAY K


Labeling Protocol (product # 208033, 208034, 208035)

Before starting the experimentTotal RNA should be prepared using a method that preserves small RNA species . When using commercially available kits, please verify that the total RNA preparation contains small molecular weight RNAs . We recommend using the miRCURY™ RNA Isolation Kits for total RNA preparations . For labeling, we recommend that you use miRCURY LNA™ microRNA Hi-Power Labeling Kit or miRCURY LNA™ microRNA Power Labeling Kit . Please visit www .exiqon .com to learn more about these products .

The amount of total RNA to be labeled for an array hybridization depends on the microRNA content of the cells or tissue being analyzed . Without prior knowledge of microRNA content in the sample, we recommend using between 250 ng and 1 µg of total RNA per labeling reaction .

Prior to performing the labeling, dissolve the fluorescent dye(s) by adding 29 µL of nuclease-free water to the tube with the labeling dye, followed by vortexing and a brief centrifugation to collect the content of the tube .

Important note:

Dissolve the spike-in miRNAs in 30 µL of RNAse free water supplied upon receipt . Leave the suspension on ice for 30 minutes to dissolve . Vortex and then spin to collect tube contents . Store the dissolved Spike-in miRNA at –20° C until use and avoid repeated cycles of freeze/thawing . You may wish to aliquot the dissolved spike-in miRNAs to avoid repeated freeze/thawing . For long-term storage, keep the vial at –80° C .

See tip 1

Minor lot variations of the labeling dyes may occur . For optimal performance ofdual color hybidization with Hy3™ and Hy5™ labeled RNA, it is recommended to use matched pairs of Hy3™ and Hy5™ labels . The miRCURY LNA™ microRNA Array, Hi-Power labeling kit (product # 208035) and miRCURY LNA™ microRNA Array, Power labeling kit (product # 208032-A) always contain matching pairs of dyes .

16

mIC

Ro

RN

A AR

RAY K


Important note:

If both the Spike-in miRNA kit v2 (supplied with the arrays) and the Spike-in miRNA kit (Product # 208040) are to be used in the labeling reaction, it is recommended to dissolve both tubes in 15µL RNase free water . Leave the suspensions on ice for 30 minutes to dissolve . Vortex and then spin to collect tube contents . Finally mix the two in one tube and store at –20° C until use and avoid repeated cycles of freeze/thawing . You may wish to aliquot the dissolved spike-in miRNAs to avoid repeated freeze/thawing . For the labeling reactions, 1µL of this joint spike-in mix is used instead of 1 µL Spike-in miRNA kit v2 .

17

mIC

Ro

RN

A AR

RAY K


Protocol & Notes

Tota

l han

dlin

g ti

me:

1 h

our

Protocol

Step 1 Thaw all kitcomponents

Place all kit components on ice and thaw for 15-20 min . Mix thoroughly by vortexing followed by brief centrifugation . Do not thaw or vortex the enzymes . Flick these tubes followed by brief centrifugation .

Step 3Incubate 30 min . at 37° C

Incubate 30 min . at 37° C, using a PCR cycler with heated lid .

Step 4Incubate 5 min . at 95° C

Stop the enzyme reaction and denaturate the RNA by incubation at 95° C followed by snap cooling on ice .

Step 2 Combine reagentsaccording to Table 2 . Mix on ice

Reagents should be combined in an RNase-free microcentrifuge tube and should be mixed by pipetting up and down to ensure that all reagents are mixed thoroughly .

** We recommend to use between 0 .25 - 1 µg, depending on the miRNA content in the sample . Mastermix preparation: In order to minimize variation between slides it is recommended to prepare master mixes for both the CIP reaction (Table 2 ) and the labeling reaction (Table 3 ) .

Table 2 Volume (µL)

Total RNA** 3

Spike-in miRNA kit v2 1

CIP buffer 0 .5

CIP enzyme 0 .5

18

mIC

Ro

RN

A AR

RAY K


Step 7 Mix and centrifuge the reagents briefly

Reagents should be mixed by gentle vortexing or by pipetting up and down to ensure that all reagents are mixed thoroughly .

Step 9 Incubate for 15 min . at 65° C

After stopping the labeling procedure, briefly spin the reaction and leave it at 4° C . The labeled sample is now ready for hybridization on the array . Hybridization should preferably occur within 1-2 h .

Step 6 Combine reagents listedin Table 3 . Mix on ice

Add the reagents listed in Table 3 to the 5 µL CIP reaction from step 5 .

Mastermix preparation: In order to minimize variation between slides it is recommended to prepare master mixes for both the CIP reaction (Table 2) and the labeling reaction (Table3 ) .

Table 3 Volume (µL)

CIP reaction from step 5 5

Hi-Power Labeling buffer 3

Fluorescent label (Hy3™ or Hy5™) 1 .5

DMSO 2

Hi-Power Labeling enzyme 1

Step 8 Incubate 2 hours at 16° C

Incubate for 2 hours at 16° C, using a PCR cycler with heated lid . Protect the reaction from light .

Step 5 Incubate 2 min .on ice .

Leave on ice for at least 2 min . and up to 15 min . Briefly spin the reaction after incubation on ice .

19

mIC

Ro

RN

A AR

RAY K


Hybridization Protocol

Hybridization and washing using Tecan HS Pro™ hybridization stations

Before starting the experimentFor labeling, we recommend using the miRCURY LNA™ microRNA Hi-Power Labeling Kit . Please visit www .exiqon .com to learn more about this product .

The amount of total RNA to be labeled for an array hybridization dependson the microRNA content of the cells or tissue being analyzed . Without prior knowledge of microRNA content in the sample, it is recommended using between 250 ng and 1 µg of total RNA per labeling reaction .

Check the hybridization buffer for any precipitate . If necessary, warm the solution at 56° C and agitate to dissolve the precipitate completely .

Please refer to the instruction manual of your hybridization station for correct volume of buffers required to perform the hybridization .The volumes in Table 1 applies to the hybridization of 4 slides in a Tecan HS400/HS4800 hybridization station .

20

mIC

Ro

RN

A AR

RAY K


Tota

l han

dlin

g ti

me:

1 h

our

Protocol

Step 1Combine the labeled sample(s)

If running a dual color experiment, the two samples from the Hy3™ and Hy5™ labeling reactions are combined on ice .If the experiment is single color, 12 .5 µL RNAse free water is added to the labeling sample . Total volume should be 25 µL .

Step 2Add 25 µL 2x Hybridization buffer

Check for precipitation (see p . 17) in the hybridization buffer before adding 25 µL to the labeled sample(s) . Mix by vortexing and spin briefly .

Step 3Denature 2 min . at 95° C

During the incubation the target preparation should be protected from light .

See tip 4

Step 4Incubate 2 min . on ice

Leave on ice for at least 2 min . and up to 15 min . Briefly spin the reaction after ice incubation .

Step 5Preparation of Tecan

Load slides in hybridization chambers . Set the program for the hybridization station: Prewash the slides with wash buffer A . Temperature 56°C, Wash time: 30 sec ., Soak time: 0 sec .

Recipes for preparation of 200 mL Wash buffers

Wash buffer A Wash buffer B Wash buffer C

20x Salt buffer 20 mL 10 mL 2 mL

10% Detergent solution 4 mL -

Nuclease-free water 176 mL 190 mL 198 mL

Table 1

See tip 2

21

mIC

Ro

RN

A AR

RAY K


Step 9Two runs of wash at 56° C for 1 min . using Wash buffer A

Set the program for the hybridization station:Temperature 56° C, Wash time: 1 min ., Soak time: 1 min .

Step 10Two runs of wash at 23° C for 1 min . using Wash buffer B

Set the program for the hybridization station:Temperature 23° C, Wash time: 1 min, Soak time: 1 min .

Step 11Two runs of wash at 23° C for 1 min . using Wash buffer C

Set the program for the hybridization station: Temperature 23° C, Wash time: 1 min ., Soak time: 1 min .

Step 12Wash at 23° C for 30 sec . using Wash buffer C

Set the program for the hybridization station:Temperature 23° C, Wash time: 30 sec ., Soak time: 0 sec .

Step 13Dry slides

Set the program for the hybridization station:Slide drying for 5 min .

Step 7Inject reaction mixture

Inject the 50 µL target preparation to the hybridization station . In order to flush injection inlet, it is recommended to inject 10µL 1x diluted Hybridization buffer after target injection .

Step 8Incubate 16 hours at 56° C

Set the program for the hybridization station: 56° C and 16 hours incubation . Agitation should be set to medium, if possible .

Step 6Flush hyb chamber with 1x Hybridization buffer

The slide chamber in the hybridization station should be primed . Check the appropriate volume of the chamber in the suppliers manual and add 1x diluted Hybridization buffer . Dilute with water . (e .g . use 100 µL for a Tecan HS400/HS4800) .

See tip 5

22

mIC

Ro

RN

A AR

RAY K


Hybridization and washing using an Agilent hybridization SureHyb chamber kit and gasket slide kitFor optimal quality, it is recommended to use an automatic hybridization station, e .g . Tecan HS Pro hybridization station (see procedure atpage 19) . If a hybridization station is not available, manual hybridization can be carried out according to the protocol in this section using an Agilent hybridization SureHyb chamber kit and gasket slide kit . Please contact www .exiqon .com/contact for an alternative protocol using cover slip .

Additional required materials:Hybridization Chamber Kit - SureHyb enabled, Agilent part # G2534AHybridization Gasket Slide Kit (5) - 1 microarray per slide format, Agilent part # G2534-60003Hybridization oven with rotation (e .g . SciGene, # 400 or Agilent) .Ethanol 99% Hybridization chamber user guide (G2534-90002)

Before starting the experiment, day 1We recommend using the miRCURY LNA™ microRNA Hi-Power labeling kit for labeling of your sample(s) . Please visit www .exiqon .com to learn more about this product .

Check the hybridization buffer for any precipitate . If necessary, warm the solution at 56° C and agitate to dissolve the precipitate completely .

23

mIC

Ro

RN

A AR

RAY K


Before starting the experiment, day 2Glass staining jar/dish and Wash buffer A should be placed at 56° C before starting the experiments at day 2 .

If one or two miRCURY LNA™ microRNA Arrays are processed together in an experiment, the arrays could be washed in a 50 mL screw-top tube (e .g . Falcon) by gently inverting the tube . If three or more miRCURY LNA™ microRNA Arrays are processed in an experiment the arrays could be placed in a slide rack and washed in a glass staining jar/dish . Use appropriate volume of washing buffer to cover the slides and shake gently . The volumes in Table 2 below are required for a large glass staining dish (8 slides, Sigma-Aldrich product # S-S6016 or similar) . The following protocol is for hybridization of miRCURY LNA™ microRNA Arrays using a Agilent Hybridization chamber - SureHyb .An instructional video on how to perform the hybridization using SureHyb Chambers can be found here: www .exiqon .com/e-talk

Table 2

The volumes in this table are required for glass staining jars of 200 mL.

Recipes for preparation of Wash buffers



10% Detergent solution 12 mL - -


24

mIC

Ro

RN

A AR

RAY K


Tota

l han

dlin

g ti

me:

1 h

our

Protocol

Step 1Prepare the labeled sample(s)

Adjust the volumes of the labeled sample to 200 µL by adding nuclease free water to the labeled sample(s) (kept on ice) . If running a dual color experiment, combine the two samples from the Hy3™ and Hy5 ™ labeling reactions before adjusting the volume .

Step 2Add 200 µL 2x hybridization buffer

If there is precipitation in the Hybridization buffer, then warm the solution at 56° C and agitate to dissolve . Add 200 µL to the labeled sample(s) . Mix by vortexing and spin briefly .



See tip 4

Step 4Incubate 2 min . on ice

Leave on ice for at least 2 min . and up to 15 min . Briefly spin the reaction after ice incubation .

Step 5Add 400 µL to reservoir

Add 400 µL of the target sample mixture to the reservoir of backing gasket slides . Place the slide on top of the the backing gasket slides with the array side facing the target samples .


Clamp the array/backing slide sandwich into the SureHyb hybridization chambers and make sure all bubbles move freely . Incubate at 56° C for 16 h . in a hybridization oven with rotation (e .g . SciGene, #400) .

Step 7Place Wash buffer A at 56° C overnight

Pre-warm the glass staining jar/dish and Wash buffer A by placing them at 56° C .

See tip 2

25

mIC

Ro

RN

A AR

RAY K


Step 11 Wash briefly at room temperature in Wash buffer B

The slide is washed briefly (one plunge) in Wash buffer B (at RT) to avoid transfer of detergent to the next wash step .

Step 12 Wash 2 min . at room temperature in Wash buffer B

The slide is washed at room temperature by plunging gently for 2 min . in a new glass staining jar/dish/Falcon tube in Wash buffer B .

Step 13Wash 2 min . at room temperature in Wash buffer C

The slide is washed at room temperature by plunging gently for 2 min . in a new glass staining jar/dish/Falcon tube in Wash buffer C . Remove veryslowly from the buffer in order to let the buffer runoff .

Step 9Collect slides in Wash buffer A

Slides are placed in a submerged slide rack in a new jar with Wash buffer A at room temperature until all slides are disassembled . Make sure the slides are kept fully submerged during washing steps, and don’t let the slides dry in between steps .

Step 14Wash briefly in 99% Ethanol

Transfer the slide rack to a new staining dishwith 99% Ethanol at room temperature . Washthe slides very briefly by plunging the rack gentlyup and down in the ethanol for a few seconds .

Step 10Wash 2 min . at 56° C in Wash buffer A

Immerse the slides in the prewarmed Wash buffer A and wash slides by plungeing gently for 2 min .

Step 8Disassemble hybridization chamber

Remove array/backing slide sandwich from SureHyb hybridization chamber . Submerge the sandwich into a jar containing Wash buffer A at room temperature and separate the slides from the backing slide using a plastic forceps .

26

mIC

Ro

RN

A AR

RAY K


Step 15Dry the slide(s)

Dry the slides by centrifugation for 5 minutes at1000 rpm . At this stage the fluorophores on theslides are very susceptible to degradation by ozone in atmospheric air . Keep ozone-induced photo bleaching to a minimum by working in an ozone free lab or keeping the slides under a controlled atmosphere . Scan slides immediately after drying .

See tip 6

Hybridization and washing using NimbleGen/ MAUI® 4-Bay or 12-Bay hybridization stationsThis Protocol provides information for the use of Exiqon miRCURY LNA™ Arrays with the NimbleGen/ MAUI® Hybridization System using a NimbleGen HX1 mixer . The protocol is designed for the arrays that are printed on the opposite side of the slide compared to the barcode (prod . # 208420, 208421 and 208422) .

The NimbleGen/ MAUI® Hybridization System is comprised of two main components, the disposable NimbleGen Mixer hybridization chambers and NimbleGen/ MAUI® instrument that powers the mixing bladders in the Mixer and maintains a constant incubation temperature . The NimbleGen Mixer adheres to the microarray slide via an adhesive gasket forming a uniform, low volume, sealed hybridization chamber . Once attached, the Mixer-slide is clamped into one of the heated slide bays in the base unit, where hybridization takes place . For details about using the NimbleGen/ MAUI® Hybridization System please see the User’s Guide that come with the Hybridization System .

27

mIC

Ro

RN

A AR

RAY K


Additional required materials:RNA samples (labeled with miRCURY LNA™ Hi-Power Labeling Kit)NimbleGen HX1-mixersNimbleGen Precision mixer alignment tool (PMAT)NimbleGen Mixer Disassembly Tool NimbleGen Gasket brayerPositive Displacement Pipette (optional, but highly recommended)Wide pipette tips (see filling video at www .exiqon .com)7 Rectangular Staining Dishes, 250 mL, w/slide washing racks (e .g . Wheaton # 900200 /VWR# 25461-003) .Heating block set to 56° C .Oven set to 56° C .

Before starting the experiment, day 1We recommend using the miRCURY LNA™ microRNA Hi-Power Labeling Kit for labeling of your sample(s) . Please visit www .exiqon .com to learn more about this product .

Check the hybridization buffer for any precipitate . If necessary, warm the solution at 56° C and agitate to dissolve the precipitate completely prepare the wash buffers and leave min . 500 mL Wash Buffer A in an oven at 56° C over night . Leave 2 Rectangular Staining Dishes in the oven at 56° C as well .

Table 2

The volumes in this table are required for glass staining jars of 200 mL.

Recipes for preparation of Wash buffers



10% Detergent solution 12 mL - -


28

mIC

Ro

RN

A AR

RAY K


Protocol

Step 2Prepare the labeled sample(s)

If running a dual color experiment, the two samples from the Hy3™ and Hy5™ labeling reactions are combined on ice . If the experiment is single color, 12 .5 µL RNase free water is added to the labeled sample . Total volume should be 25 µL .

Step 1Attach the NimbleGen HX1-mixer to the miRCURY LNA™ Array

For details on how to assemble the mixer and the array slide please see the NimbleGen User’s Guide . Briefly:• Remove the miRCURY LNA™ Array

from the slide box .• Insert the array slide in the PMAT with the

barcode facing down and away from the hinge .

• Place the HX1 mixer in the lid of the PMAT with the end of the mixer towards the hinge and the mixer’s adhesive gasket facing outward . Remove the protective lining from the mixer using a forceps and close the lid of the PMAT and carefully press the mixer .

• Remove the mixer-slide assembly from the PMAT and place the assembly with the mixer side up on a heating block at 56° C for 5 min .

• Use the NimbleGen Gasket brayer and moderate pressure to ensure good mixer to slide adhesion .

• Leave the mixer-slide assembly with the mixer side upon the heating block at 56° C . The sample must be loaded onto the 56° C-heated slide within 30 min of assembly .

See tip 2

29

mIC

Ro

RN

A AR

RAY K


Step 3Add 25 µL 2X Hybridization buffer

Add 25 µL 2X Hybridization buffer to the labeledsample(s) . Mix by vortexing and spin briefly .Final volume 50 µL .



Step 5Cool 2 min . on ice

Leave on ice for at least 2 min . and up to 15 min . Briefly spin the reaction after ice incubation . Heat the sample to 56° C for 2 min . prior to loading into the slide-mixer assembly .

Step 6Load the sample into the pre-heated HX1-mixer-slide assembly

Briefly spin the pre-heated sample prior toopening the tube . Aspirate 45 µL of sampleinto the pipette by pipetting up and down afew times (avoid bubbles) .

See tip 3

30

mIC

Ro

RN

A AR

RAY K


Step 9Prepare wash buffers

At room temperature prepare the following washing solutions, each in a separate staining dish . Add sufficient liquid to completely cover the slides when they are placed in a washing rack in the staining dish: Wash A, B and C buffer according to table 2 . Ethanol, 99% .


Place the loaded slide-mixer assembly in one of the bays of a NimbleGen mixer, close the lid and incubate at 56° C for 16 h with mixing mode B .

Step 6 (Continued)Load the sample into the pre-heated HX1-mixer-slide assembly .

• Insert the pipette tip into the fill port in the tab-end of the HX1-mixer and carefully inject the sample into the chamber at a moderate speed until sample emerges from the vent port (if sample is injected to slowly, a bubble might form) . The actual volume of the HX1-mixer varies slightly from batch to batch, so do not be alarmed should some of the sample bubble up from the vent port .

• Keep the plunger depressed and remove the pipette tip from the fill port . Any excess sample from the fill and vent ports are wiped clean with a tissue .

• Using forceps, place adhesive port seals directly over both ports . Use a finger on each port seal and press down firmly on both seals simultaneously to seal the ports .

Step 8Preparation of washing procedure

In preparation of next day’s washing procedure pre-heat min . 500 mL Wash Buffer A at 56° C overnight . Leave 2 Rectangular Staining Dishes in the oven at 56° C as well .

31

mIC

Ro

RN

A AR

RAY K


Step 10Disassemble HX1-mixer and slide

Perform the following operations at 56° C by working in the door opening of an oven:A . Place the mixer/slide assembly in the Mixer

Disassembly Tool in the heated staining dish and add sufficient pre-heated Wash Buffer A to cover it .

B . To the other pre-heated staining dish add sufficient Wash Buffer A to completely cover the slides when placed in the washing rack .

C. Remove the HX1-mixer from the slide and quickly insert it into the submerged washing rack to avoid cooling of the slide .

Step 13Wash 2 min . at room temperature in Wash buffer C

Transfer the slide rack to a new staining dish with Wash Buffer C at room temperature . Wash the slides for 2 minutes by plunging the rack gently up and down in the buffer . Remove very slowly from the buffer in order to let the buffer run off .

Step 12 Wash 2 min . at room temperature in Wash buffer B

When all slides have been collected in Wash Buffer B, wash the slides for additional 2 minutes by plunging the rack gently up and down in the buffer at room temperature . Make sure the slides are kept fully submerged during washing steps, and don’t let the slides dry between steps .

Step 11Wash 2 min . at 56° C in Wash buffer A

D . Wash the slide for 2 min at 56° C by gentle plunging of the slide rack .E . Transfer the slide to the rack in Wash

Buffer BF . Repeat steps C to F for each slide in the

NimbleGen Hybridization Station, collecting the slides submerged in Wash Buffer B at room temperature .

32

mIC

Ro

RN

A AR

RAY K


Step 15Dry the slide(s)

Dry the slides by centrifugation for 5 minutes at 1000 rpm . At this stage the fluorophores on the slides are very susceptible to degradation by ozone in atmospheric air . Keep ozone-induced photobleaching to a minimum by working in an ozone free lab or keeping the slides under a controlled atmosphere . Scan slides immediately after drying .

See tip 5

Step 14 Wash briefly in 99% Ethanol

Transfer the slide rack to a new staining dish with 99% Ethanol at room temperature . Wash the slides very briefly by plunging the rack gently up and down in the ethanol for a few seconds .

33

mIC

Ro

RN

A AR

RAY K


Recommendations for Experimental Set-up and Data HandlingAs mentioned earlier (page 13), it is possible to hybridize one sample (i .e . single color) or two samples (i .e . dual color) to one array . Since microarray expression profiling without appropriate standards cannot be used for absolute quantification, expression levels of a microRNA in a sample can only be determined in comparison to other samples . In single color experiments, each sample is hybridized to a separate array; the comparison must then occur between arrays . So far there are no established or thoroughly tested control or housekeeping small ncRNAs or microRNAs that can be used as common factors for normalization . The only options for single-color experiments are the use of the characteristic signal distributions, assuming that the similarity between the samples is high enough to allow normalization, or the use of synthetic spiked-in microRNAs . A set of spiked-in control microRNAs could also be an option, but again, it has to be considered that the number of spots used is limited and may thus introduce bias . A way to enable optimal normalization across arrays is to use dual-color arrays with a common reference sample or a universal reference on all arrays in the study . Once intra-slide normalization has taken place, the log2 ratios between sample and reference for each microRNA can be calculated allowing the immediate direct comparison of all log2 ratios from all slides . The fact that all microRNA signals are expressed as a ratio to a reference, which should be the same on each slide, in essence removes technical variations from the comparison .

For help on designing array experiments, an overview can be downloaded from www .exiqon .com/array: Guidelines_for_setting_up_microRNA_array_profiling_exp .pdf

Protocol

Step 1Scanning

We are using an Agilent G2505B Microarray Scanner System . The scanning is normally performed with 10 µm . The sensitivity should be adjusted to 100% PMT . To avoid ozone bleaching, we scan the microarrays in an ozone-free environment (less than 2 ppb ozone) . Before starting any analysis, confirm that the tiff image is in the correct orientation (two landing lights in lower right corner) . Depending on the scanner, the image may need to be flipped from upper left to lower right .

34

mIC

Ro

RN

A AR

RAY K


Step 2Spot evaluation and background subtraction

In general, we recommend using local background subtraction . We subtract the local median background signal from each spot using the Exiqon-tailored ImaGene data analysis software (see www .exiqon .com/ mirna-array-software), When using more advanced background subtraction, ‘Normexp plus offset’ convinced us with satisfying results . We are not using Feature Extraction software (Agilent) on a routine level . However, we provide a short protocol for customers who like to use this software (www .exiqon .com/array) .

Step 3Normalization

When running a dual color experiment we recommend a lowess intra-slide normalization for the signal intensities of each channel as a minumum . This eliminates the dye- and label-specific variances . In addition, it is recommended to monitor inter-slide comparability based on the spike-ins and or signals derived from constantly expressed RNAs . If running single color experiments we recommend to normalize the data using the quantile normalization method, as we have found that this generates the most reliable data .Both of these normalization methods are supported in the Nexus software, part of the (the Exiqon array data analysis supplied software package, for details see www .exiqon .com/mirna-array-software)miRCURY LNA™ microRNA Arrays contain several control capture probes (e .g . detecting U6 snRNA and snoRNAs) and the signal obtained from these probes could theoretically be used in normalization after confirming the constant expression of these small RNAs under the given experimental conditions . However, we believe that normalization based on these very few probes alone is not optimal . Therefore, we recommend using these control capture probes to monitor the analyzed samples for uniformity and not for normalization .

35

mIC

Ro

RN

A AR

RAY K


Step 3 (Continued) In theory, it is possible to use signals from a set of spike-in synthetic microRNAs (added to each labeling reaction and for which control capture probes exist) to perform normalization . However, apart from being something synthetic added to the samples, the use of spike-ins for normalization focuses on a small number of data points, which is a problem if the differences between the samples are very large or if something in the samples themselves affect the synthetic microRNAs during labeling or hybridization . Exiqon offers two different spike-in kits . The one supplied with the array (spike-in miRNA kit v2, 208041), contains 52 synthetic miRNAs and could in theory be used for normalization, although we recommend using all detection probes instead . The other (spike-in kit, 208040) only contains 10 different miRNAs and can be used for control of the hybridization (see page 8), while it is recommended not to be used for normalization .

Step 4Data analysis and visualization

From each spot and each channel the median signal intensity obtained after image analysis should be measured and normalized (after either local background subtraction or normexp pluss offset background subtraction) . The difference of a normalized and an unnormalized dataset can be seen in the MA plots below . For each of the normalized four replica datasets, the ratio between the Hy3 and Hy5 channels is determined .The way the actual comparison is performed depends on the experimental setup . For direct comparison, the log ratios can be used directly . For common /universal reference comparisons log2 differences between sample are compared indirectly between the slides by using the common reference as normalizer added up to obtain the difference between the samples . We recommend using the special tailored Exiqon-offered Nexus software (see www .exiqon .com/mirna-array-software), that calculates significant differentially expressed miRs across samples and visualizes this in heatmaps/cluster diagrams .

36

mIC

Ro

RN

A AR

RAY K


Step 4 (Continued)

Step 5Data evaluation

We strongly advise users to evaluate the microRNA data for their cluster and family performance . MicroRNAs which cluster in close proximity are expected to react similarly in their expression pattern, due to common transcriptional activity . MicroRNA families can be interesting to analyze since they may react similarly due to their common target sequences and help understanding how family members are tissue-specifically regulated . An analysis of how the data of families or clusters correlate can therefore provide relevant data in addition to the actual microRNA signal of initial interest . Additionally, a further analysis of potentially regulated mRNAs targets will be useful . A short list of useful software and databases can be found below .

10

5

0

-5

-10

0 5 10 15

M

10

5

0

-5

-10

0 5 10 15

Two color intra-slide mA-plots obtained before (left) and after (right) lowess intra-slide normalization. Colored spots represent spike-ins of different signal intensities.

37

mIC

Ro

RN

A AR

RAY K


Software and Databases

Exiqon offers a software package tailored to suit the needs of analysis of the miRCURY LNA™ microRNA Arrays . The easy-to-use software package includes ImaGene® 9 for image analysis and Nexus Expression™ 2 for array data analysis . Together with the Exiqon specific settings file and the Exiqon quick manual, a successful array analysis can be obtained with a few clicks . Brief descriptions of the two software tools are given below . Further information is available at www .exiqon .com/mirna-array-software .

ImaGeneThe Exiqon-offered image analysis software ImaGene® 9 places a grid on top of the scanned array image, and identifies which probe is located in each spot . The software quantifies the signal intensity and the surrounding background . ImaGene® 9 can also be used for basic data normalization . ImaGene in our hands outperforms other software regarding precision in spot recognition and flagging .

Nexus ExpressionNexus Expression™ 2 is the miRCURY LNA™ microRNA Array supportive software for statistical analysis of array profiling data . Nexus Expression™ 2 is fully compatible with ImaGene® but also other common image analysis program output formats are supported . Nexus Expression™ 2 allows background subtraction, normalization and visualization of array data . It can combine the replicated measures of each probe on the arrays into one output value per array and make statistical calculations of the differentially expressed data obtained by comparing the microRNA signal data of different array experiments . The active links of the probe-target information to miRBase allows for easy retrieval of further microRNA information . Additional information can be found on the Exiqon website at www .exiqon .com/mirna-array-software .

Several other commercial and free software packages for microarray image and data analysis are also available . A selection of these is listed below . The list is not complete and a more appropriate solution may be available for certain projects . For a more comprehensive overview of software packages, it is advised to visit statweb at www .statsci .org/micrarra/index .html .

38

mIC

Ro

RN

A AR

RAY K


Links to other array software:Image analysis:Bzscan . . . . . . . . . . . . . . . . . . . . http://tagc .univ-mrs .fr/ComputationalBiology/bzscan/ScanAlyze . . . . . . . . . . . . . . . http://rana .lbl .gov/EisenSoftware .htmSpotfinder . . . . . . . . . . . . . . . http://www .tm4 .org/spotfinder .html

Statistical analysis:Carmaweb . . . . . . . . . . . . . . https://carmaweb .genome .tugraz .at/DChip . . . . . . . . . . . . . . . . . . . . . . https://sites .google .com/site/dchipsoft/downloading-dchip-softwareMidas . . . . . . . . . . . . . . . . . . . . . . http://www .tm4 .org/midas .htmlR/bioconductor / limma . . . . . . . . . . . . . . . . . . . . . . http://www .r-project .org/ http://www .bioconductor .org/ http://bioinf .wehi .edu .au/limma/

Links to additional array analysis software and microRNA software and databases in general are available at www .exiqon .com/array

39

mIC

Ro

RN

A AR

RAY K


Experimental procedure

Preparation of RNA sampleTotal RNA should be prepared using a method that retains small RNA species . When using commercially available kits, please ensure that the total RNA preparation is guaranteed to contain microRNAs .We recommend using our miRCURY™ RNA Isolation Kits for total RNA puri-fication . Procedures that include acidic phenol chloroform extraction are generally also recognized as methods that preserve small RNAs . However, we recommend a column purification step following the phenol:chloroform extraction to remove any trace of these chemicals, as they could potentially inhibit the labeling reaction . Our miRCURY™ RNA Isolation Kits can be used for this procedure .

The purified total RNA should be dissolved in RNAse-free water or TE buffer at a concentration of no more than 2 µg/µL . It is recommended to assess the integrity of the RNA isolated before proceed ing with labeling . This may be performed either on the Agilent Bioanalyzer (RIN values should be above 7) or by denaturing gel electrophoresis . Degraded RNA is generally not suitable for labeling or for hybridization to microarrays . However, RNA extracted from FFPE samples does give good results on miRCURY LNA arrays . The miRCURY LNA™ microRNA Hi-Power Labeling Kit can be used for efficient labeling of both microRNA enriched and total RNA . However, microRNAs constitute only a small fraction (~0 .01%) of the total RNA . Attempts to purify this small fraction can result in loss of microRNAs or co-purification of larger RNA species . For this reason, we recommend using total RNA for labeling and hybridization .The amount of total RNA to be labeled for an array hybridization depends on the microRNA content of the cells or tissue being analyzed . Without prior knowledge of the microRNA content we recommend using between 250 ng and 1 µg of total RNA per labeling reaction .

Tip 1

Tips and Trouble Shooting

40

mIC

Ro

RN

A AR

RAY K


Black spots - purification of the labeling reaction IGhost spots can be avoided by removing unincorporated dye . We recommendthat the labeled RNA is purified using miRCURY™ RNA Isolation Kit or an exclusion column e .g . Roche mini Quick Spin™ OLIGO columns or BioRad Micro Bio-Spin columns . Alternatively, perform the ethanol precipitation shown below:• Mix the Hy3™ and Hy5™ labeling reactions before precipitation .• Add 2 .5µL RNase free Sodium acetate (3M, pH5 .5) to the 25µL labeling

reaction and 75µL 99 .9% ethanol .• Incubate the sample at -20° C for 30 minutes . • Centrifuge 30 minutes at max speed in a cooled centrifuge .• Remove supernatant and wash with 80% precooled ethanol, by centrifuging

5 min at max speed in a cooled centrifuge .• Remove supernatant and if necessary speedvac for a few minutes to

remove remainder of ethanol .• Dissolve in a mixture of 4µL DMSO, 15µL water and 6µL labeling buffer .

Concentration and purification of the labeling reaction IIWe recommend that you follow the standard instruction manual for the labeling reaction, which will yield 50 µL (if running dual color) . To minimize the number of handling steps through which the concentration of microRNAs may be compromised, we recommend loading 45 µL of the sample into the NimbleGen HX1 mixer without a concentration/purification step; However if high fluorescent background is repeatedly observed, you may purify the labeling reactions to remove unincorporated dyes . For purification and/or concentration of the sample we recommend miRCURY™ RNA Isolation Kits, alternatively either ethanol precipitation or the RNeasy Mini Kit . Due to the bleaching effect of ozone on Hy5™ it is important to finish the concentration of the labeling reaction in the shortest amount of time possible .

Ethanol precipitation:The combined labeling reaction can be concentrated by either ethanol precipitation as shown below, or as described in Tip 1:• Add 1/10 vol RNase free sodium acetate (3M, PH 5 .5) to the 25 µl labeling

reaction + 3 vol 100% ethanol

Tip 2

Tip 3

41

mIC

Ro

RN

A AR

RAY K


• Incubate the sample at –20° C for 20 minutes • Centrifuge 20 minutes at > 12000g at 4° C • Remove supernatant and wash the pellet with 200 µl 80% precooled ethanol

(-20° C)• Centrifuge 5 minutes at > 12000g at 4° C• Remove supernatant and let the pellet dry for 5 minutes• Dissolve in a mixture of 20 µl: 3 .2 µl DMSO, 12 µl H2O and 4 .8 µl labeling

buffer

miRCURY™ RNA Isolation Kits• Please refer to the miRCURY™ RNA Isolation Kit manual for an RNA

concentration protocol .

Solid particlesIf you are concerned about introducing microscopic solid particles onto your array, then filter the sample through a Millipore 0 .22 Ultrafree-MC Centrifugal Filter (product # UFC30GV0S): Wet the filter with 20 µL Nuclease-free water, spin 1 min . at 12,000 rpm and remove water . Add the target preparation and repeat the centrifugation . The flow-through contains the labeled sample(s) .

Flushing the hybridization chamberIn order not to lose any target when using automated hybridization stations, it is recommended that you inject a volume smaller than the total volume of the hybridization chamber . The mixing mechanism of the hybridization station will ensure that the injected sample will be distributed equally across the entire array . To ensure that the composition of the hybridization buffer is the same after mixing it is recommended to flush the hybridization chamber with 1X hybridization buffer immediately prior to sample injection .

Dry slidesIf you are doing manual hybridization and have more than 2 slides in your experiment you can dry the slides in a centrifuge by placing the slides in a slide rack on a swinging plate tray (1,000 rpm for 5 minutes) . Alternatively, place your slides back to back in a screw-top tube and spin at 1,000 rpm for 5 minutes in a centrifuge .

Tip 4

Tip 5

Tip 6

42

mIC

Ro

RN

A AR

RAY K


No signalsCheck that the Hy3™ labeled “landing lights” are visible . They are located in all 4 corners plus one extra in the lower right corner of the 48 sub-arrays, 240 total . If they are visible, please check that signals from the spike-in controls used in the labeling can be seen . If not, the labeling procedure probably has failed . If the spike-in controls can be seen, please check that your total RNA sample is of good quality by gel electrophoresis and optical density analysis . If the RNA quality is good, then try to increase the amount of RNA used in the labeling . If signals in the Hy5™ channel are unexpectedly low, it could be due to high ozone levels in the air . Ozone has a bleaching effect on the Hy5™ . Exiqon recommends to perform labeling reaction, slide handling and scanning in an ozone free environment .

High signalsDue to high binding affinity of the LNA™-enriched miRCURY™ capture probes, it is of utmost importance to use high stringency experimental settings, i .e . using the miRCURY LNA™ microRNA Array hybridization buffer and an overnight hybridization temperature of 56° C . Furthermore, use of ½-1 µg total RNA will in most cases result in optimal array signal intensities .

High backgroundUsing a manual hybridization procedure with cover slip may produce high background around the margins of the coverslip . This is usually caused by evaporation of the hybridization solution . To avoid uneven distribution of the hybridization solution, it is important to position the slide horizontally . To increase the humidity, we recommend using a water bath .

Tip 7

Tip 8

Tip 9

43

mIC

Ro

RN

A AR

RAY K


Spike-in miRNA signal distribution

Sample signal distribution for Spike-in miRNA Kit v2 (product # 208041)Figure 3 below shows the distribution of the 52 spike-in microRNAs spiked into 0 .25µg universal reference RNA (Ambion, AM6000) . The concentration of the various spike-in microRNAs are optimized such that the signal intensities of these spike-in microRNAs are in the dynamic range of naturally expressed microRNAs in most tissues .

Tip 10

100000

10000

1000

100

1010 100 1000 10000 100000

Hy3

™ s

igna

l slid

e 2

Hy3™ signal slide 1

miRNAsspike-in kit v2

Figure 3. Scatter plot of two hybridizations with spike-in miRNA kit v2 added . One µL of the Spike-in microRNA kit v2 was spiked into each sample of 0 .25 µg total RNA from a mix of human tissues and labeled with Hy3™ . Labeling was performed using the miRCURY LNA™ microRNA Power Labeling Kit . Hybridization was performed using the Tecan HS4800™ Pro hybridization station .

Figure 3

44

mIC

Ro

RN

A AR

RAY K


Sample signal distribution for Spike-in miRNA Kit (product # 208040)Figure 4 below shows the distribution of the 10 spike-in microRNAs spiked into 1 µg of total RNA from human lung samples . The concentration of the various spike-in microRNAs are optimized so the signal intensities of these spike-in microRNAs are in the dynamic range of naturally expressed microRNAs in most tissues .

Figure 4

The position of signals from the spike-in microRNA set compared to signals from microRNAs will depend upon the microRNA expression level in the sample .

Figure 4. Scatter plot of a self-self hybridization with spike-in mix . One µL of the Spike-in miRNA kit was spiked into a sample of 1 µg total RNA from human lung labeled with Hy3™ . Another 1 µL of spike-in microRNAs were spiked into 1 µg RNA from human lung and labeled with Hy5™ . Labeling was performed using the miRCURY LNA™ microRNA Power Labeling Kit .Hybridization was performed using the Tecan HS4800™ Pro hybridization station .

100000

10000

1000

100

1010 100 1000 10000 100000

Hy5

™ s

igna

l

Hy3™ signal

miRNAsspike-ins Spike_controls j, i, d and h

Spike_controls g, e and c

Spike_controls f, b and a

Tip 11

45

mIC

Ro

RN

A AR

RAY K


References

• The microRNA Registry . Griffiths-Jones S . Nucleic Acid Research, 2004, 32, Database Issue, D109-11

• miRBase, http://www .mirbase .org

• www .exiqon .com/array

46

mIC

Ro

RN

A AR

RAY K


Related Products

Exiqon offers a range of tools designed for microRNA isolation, expressionanalysis, localization and functional analysis .

Figure 5

miRCURY™ RNA Isolation KitsGet high quality total RNA suitable for miRCURY LNA™ microRNA Array analysis in as little as 20 minutes . Protocols are available for a large number of sample types and organisms .

miRCURY LNA™ microRNA Hi-Power Labeling KitFor fluorescent labeling of microRNAs from total RNA samples ready for array hybridization (product # 208033, 208034, 208035) .

miRCURY LNA™ microRNA Array, ready-to-spot probe setReady-to-spot oligo for direct printing of arrays, or coupling in bead-based applications (product # 208410-A) .

miRCURY LNA™ microRNA Array, Spike-in miRNA kitTen different synthetic unlabeled microRNAs in different concentrations . The spike-in microRNA kit will hybridize to corresponding capture probes on the miRCURY LNA™ microRNA Array (product # 208040) .

miRCURY LNA™ microRNA Array, Spike-in miRNA kit v252 different synthetic unlabeled microRNAs in different concentrations . The microRNAs wll hybridize capture probes on the miRCURY LNA™ microRNA Array (product # 208041) .

Functional AnalysisLocalizationExpression

AnalysisIsolation

47

mIC

Ro

RN

A AR

RAY K


miRCURY LNA™ microRNA Array, Hybridization buffer5 mL hybridization buffer optimal for microRNA hybridization to the miRCURY LNA™ microRNA Arrays (product # 208022) .

miRCURY LNA™ microRNA Array, Wash buffer kit125 mL salt buffer and 15 mL detergent optimal for wash of miRCURY LNA™ microRNA Arrays . (product # 208021) .

miRCURY LNA™ microRNA DetectionFor in situ hybridization and northern blotting of all annotated microRNAs . miRCURY LNA™ microRNA ISH Buffer Set (FFPE) .

miRCURY LNA™ microRNA ISH optimization kit (FFPE)Complete kit with control probes and hybridization buffer for easy set upof microRNA in situ hybridization .

miRCURY LNA™ microRNA Inhibitors and Power InhibitorsUnravel the function of microRNAs by microRNA inhibition . Sophisticated LNA™ design ensures potent inhibition of all microRNAs regardless of their GC content . Chemically modified, highly stable Power Inhibitors for unrivalled potency .

miRCURY LNA™ microRNA Inhibitor LibraryFor genome-wide high throughput screening of microRNA function .

miRCURY LNA™ Universal RT microRNA PCR Exiqons microRNA qPCR system offers the best available combination of performance and ease-of-use on the microRNA real-time PCR market . The combination of a Universal RT reaction and LNA™-enhanced PCR primers results in unmatched sensitivity and specificity . The Ready-to-use microRNA PCR panels enable fast and easy microRNA expression profiling .

48

mIC

Ro

RN

A AR

RAY K


© Copyright 2011 Exiqon . All rights reserved

Literature citations:Please refer to miRCURY LNA™ microRNA Array when describing a procedure for publication using this product .

Patents and TrademarksExiqon, LNA™, Hy3, Hy5, miRPlus and miRCURY™ are registered trademarks of Exiqon A/S, Vedbaek, Denmark . Locked Nucleic Acids (LNA™) are covered by patents and patent applications owned by Exiqon A/S . ImaGene® and Nexus Expression™ are registered trademarks of BioDiscovery, Inc . All other trademarks are the property of their respective owners .

DisclaimerProducts are for research use only and not for diagnostic or therapeutic use . The products in their original or any modified form may be used only for the buyer’s internal research purposes and not for commercial, diagnostic, therapeutic, or other use, including contract research . The buyer may not resell products in their original or any modified form . The purchase of products does not include or carry an implied right or license for the buyer to use such products in the provision of services to third parties and a license must be obtained directly from Exiqon A/S for such use .This product and its use are covered by one or more of the following patents owned by Oxford Gene Technology Limited or Oxford Gene Technology IP Limited: US 6,054,270, US 5,700,637, EP 0,373,203; Jap . 3,393, 528 and 3,386,391 and pending patents . The purchaser is licensed to practice methods and processes covered by these patents using this product for its own internal research purposes only but may not: transfer data derived from the use of this product to third parties for value; use this product in the provision of services to third parties for value; use this product to make, have made, create or contribute to the creation of stand alone expression database products for license, sale or other transfer to a third party for value; or use this product for the identification of antisense reagents or the empirical design of probes or sets of probes for using or making nucleic acid arrays . The products are for research use only and not for diagnostic or therapeutic use . Specifications in this document are subject to change without notice . The array is guaranteed to cover a minimum of 98 % of the miRBase version that it was designed for .

49

mIC

Ro

RN

A AR

RAY K


outside North AmericaExiqon A/S · Skelstedet 16DK-2950 Vedbaek · DenmarkPhone +45 45 660 888Fax +45 45 661 888

North AmericaExiqon Inc . · 14 F Gill StreetWoburn, MA 01801 · United StatesPhone +1 781 376 4150Fax +1 781 376 4152 Toll free (US) +1 888 miRCURY

www .exiqon .com/contact www .exiqon .com

13-0

072

- 91

8550

- v

2 .1-

03/

2011

Documents

miRCURY LNA™ microRNA Array Kit - Exiqon · Hybridization and washing using an Agilent hybridization SureHyb ... miRCURY LNA™ microRNA Power Labeling Kit or miRCURY LNA™ microRNA